Vaccines against an oncogenic isoform of esr1 and methods of using the same

ABSTRACT

Methods of reducing the likelihood of a cancer or precancer developing resistance to a cancer therapeutic or prevention agent are provided herein. The methods include administering the cancer therapeutic or prevention agent and a vaccine comprising a polynucleotide encoding a polypeptide whose expression or activation is correlated with development of resistance of the cancer or precancer to the cancer therapeutic or prevention agent to a subject. The vaccine may include a polynucleotide encoding an ESR1 polypeptide or a truncation, deletion or substitution mutant thereof. Methods of using the vaccine including the polynucleotide encoding the ESR1 polypeptide to treat a cancer or precancer are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 62/021,586, filed Jul. 7, 2014, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded by the Department of Defense grant number BC113107. The United States has certain rights in this invention.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is incorporated herein by reference in its entirety. The Sequence Listing was filed with the application as a text file on Jul. 7, 2015.

INTRODUCTION

This application relates to a cancer vaccine against ESR1, specifically a vaccine against ESR1 isoform antigens that are expressed on cancer cells or in response to development of resistance to a therapeutic intervention to cancer (or pre-cancers). Methods of using the vaccines are also provided.

Cancer vaccines target antigens expressed by tumors, but application of these vaccines has not been as effective as once hoped due to induction of immune tolerance by chronic overexpression of the targeted protein in the absence of co-stimulatory molecules and the induction of an immunomodulatory environment. Preventative cancer vaccines may be more promising, but cancers are highly variable, with multiple genetic changes, but few truly universal changes. Thus, it is difficult to predict what antigens will be overexpressed on any specific cancer or whether an individual should be vaccinated and if so, with what antigens. In contrast, a strategy is proposed here in which vaccination against the antigen(s) that will predictably be overexpressed in response to a therapy, but prior to that antigen's over-expression by the cancer cells is used to induce a robust anti-cancer immune response.

SUMMARY

Provided herein is a mechanism of revolutionizing cancer therapy or prevention by preventing the development of resistance to cancer therapeutic or cancer prevention agents by identifying which antigens are likely to be expressed in a cancer or precancer in response to treatment with a cancer therapeutic or prevention agent and thus which antigens may be targeted with a vaccine in patients.

A vaccine targeting a specific antigen involved in a resistance mechanism, namely ESR1, and methods of using the vaccine are provided. In one aspect, the vaccine includes a polynucleotide encoding an ESR1 polypeptide, a mutant of an ESR1 polypeptide, or a portion of an ESR1 polypeptide. For example, ESR1 polypeptides of SEQ ID NO: 1-3 or 5 or portions thereof may be included in a vaccine as detailed below. SEQ ID NO: 1-3 and 5 each provide single amino acid substitution mutants of ESR1. SEQ ID NO: 1 is an ESR1 polypeptide with an Y537N mutation. SEQ ID NO: 2 is an ESR1 polypeptide with a Y537S mutation. SEQ ID NO: 3 is an ESR1 polypeptide with a D538G mutation. SEQ ID NO: 5 is an ESR1 polypeptide with a K303R mutation.

In another aspect, methods of treating a cancer or precancer or reducing the likelihood of the cancer or precancer to develop resistance to a cancer therapeutic or prevention agent by administering the vaccine provided herein to a subject with cancer or precancer are provided. The vaccine may be administered before, concurrently with or after administration of the cancer therapeutic or prevention agent. The vaccine may also be administered before, concurrently with or after administration of checkpoint inhibitory immunomodulatory agents such as antagonistic antibodies specific for CTLA-4 or PD1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that ESR1 mutants can confer ER Signaling in the absence of estrogen stimulation. 293T cells stably expressing the indicated genes were transfected with an ERE luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 2 is a graph showing analysis of ESR1-Y537N mutant signaling. 293T cells stably expressing the indicated genes were reverse transfected with 45 different pathway reporter luciferase vectors (180 ng/well), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 3 is a graph showing analysis of ESR1-K303R mutant signaling. 293T cells stably expressing the indicated genes were reverse transfected with 45 different pathway reporter luciferase vectors (180 ng/well), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 4 is a graph showing ESR1 PR signaling with different mutants. 293T cells stably expressing the indicated genes were transfected with an PR luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 5 is a graph showing ESR1 RAR signaling with different mutants. 293T cells stably expressing the indicated genes were transfected with an RAR luciferase reporter (180 ng), along with a LacZ control (20 ng) and plated in 96 well plates (20,000 cells per well). At 24 hours post-transfection, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 6 is a set of figures showing that ESR1-mutants can confer estrogen-independent growth in ER+ breast cancer in vivo. Stably infected MCF-7 cells were injected into the flank of SCID-Beige male mice (or female where indicated) at a concentration of 1M cells in 100 μl of PBS at Day 0. In female control group, a 17B-Estradiol pellet (60 day release, Innovative Research of America, Sarasota Fla.) was also implanted. Tumor growth was then monitored as indicated by caliper measurement (N=5 or 10 per group, bars represent SE) and is shown in the graph (FIG. 6A) and reported in the table (FIG. 6B). The photograph at the bottom right of the figure (FIG. 6C) is from a previous experiment measuring the growth of MCF-7-ESR1-Y537N cells in female mice supplemented with 17B-Estradiol pellets (top) versus male mice without supplementation (bottom).

FIG. 7 is a graph showing ESR1 ERE signaling in murine mammary cells with different mutants. MM3MG cells stably expressing the indicated genes were transfected with an ERE luciferase reporter (180 ng), along with a LacZ control (20 ng) using FugeneHD. At 24 hours post-transfection, cells were split and plated into 96 well plates (20,000 cells per well) where they were treated with or without 17B-Estradiol (20 nM) as indicated. 24 hours post-treatment, cells were lysed and luciferase activity measured (normalized to LacZ transfection control). N=4 per group.

FIG. 8 is a graph showing that ESR1 mutants in murine mammary cells do not confer proliferative advantage. MM3MG cells stably expressing the indicated genes were plated in 96-well plates (5,000 per well) and assessed at 4 days post-plating by MTT Assay (N=12, bars indicate SD).

FIG. 9 is a set of figures showing that ESR1 mutants in murine mammary cells can confer an advantage in anchorage-independent growth. MM3MG cells stably expressing the indicated genes were plated in 12-well dishes plates (2,500 per well) and assessed at 3 weeks days post-plating at 4× and 10× magnification (N=4, bars indicate SD). FIG. 9A is a graph showing the number of colonies per well for cells expressing the indicated ESR1 protein and either exposed to estrogen or not. FIG. 9B is a set of photographs showing anchorage independent cell growth of the indicated cells.

FIG. 10 is a graph showing ESR1 mutants in murine mammary cells can confer a proliferative advantage in vivo. MM3MG cells stably expressing the indicated genes were implanted subcutaneously into SCID-Beige mice (100,000 per mouse in PBS) at days 0. Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE).

FIG. 11 is a graph showing that the adenoviral vaccines targeting ESR1 elicit significant T-cell responses against ESR1-specific epitopes. C57 mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and sacrificed at 2 wpi. ELISPOT assays were then performed using 500 k splenocytes per well against the indicated antigen stimuli (N=5, bars represent SD).

FIG. 12 is a graph showing that the adenoviral vaccines targeting ESR1 elicit significant B-cell responses against ESR1-specific epitopes. C57 mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and sacrificed at 2 wpi. ELISA assays were then performed using ESR1 coated plates (Thermo Fisher, 10 μg/ml) and using an anti-mouse IgG-HRP secondary antibody (CST, 1:1000 dilution) to detect ESR1-specific IgG antibodies. (N=5, bars represent SD).

FIG. 13 is a graph showing that targeted vaccination against ESR1 mutants suppresses the growth of ESR1-mutant expressing cells. BALB/c mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and MM3MG cells stably expressing the indicated genes were implanted at 2 wpi (100,000 per mouse in PBS, indicated at day 0). Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE).

FIG. 14 is a graph showing that the adenoviral vaccines targeting ESR1 elicit T-cell responses against ESR1-specific epitopes in tumor-bearing mice. BALB/c mice were vaccinated using the indicated adenoviral vectors (2.6E10 viral particles per mouse via footpad) and MM3MG cells stably expressing the indicated genes were implanted at 2 wpi (100,000 per mouse in PBS, indicated at day 0). Tumor growth was measured by calipers at the indicated days (N=5, bars represent SE). ELISPOT assays were then performed using 500 k splenocytes per well against the indicated antigen stimuli (N=5, bars represent SD).

DETAILED DESCRIPTION

Approximately 70% of all breast cancers are classified as estrogen receptor positive (ER+); dependent upon constitutive estrogen receptor signaling⁶. Although different classes of endocrine (anti-estrogen) therapies (including selective estrogen receptor modulators (SERMS), downregulators, and aromatase inhibitors (AIs)) are effective treatments for these cancers in adjuvant settings, approximately 50% of women will eventually relapse and die from metastatic ER+ disease⁷⁻⁹. Thus, despite the advent of newer therapies (such as AIs) there remains an unrelenting rate of recurrence in ER+ breast cancer, particularly in cases where metastasis has occurred¹⁰⁻¹². Significantly, all patients that develop metastatic ER+ disease will progress to an endocrine therapy resistant disease. At this stage, there is no cure for ER+ breast cancer. Because compensatory mechanisms appear to account for resistance that develops in a significant percentage of anti-estrogen treated patients, we propose a novel approach that has the potential to target critical driver mutations for the lifetime of the patient. Provided herein is specifically targeted immunotherapy directed toward specific resistance drivers that are predictably evoked by compensatory resistance mechanisms.

As a novel alternative to vaccines targeting well established tumor antigens, we hypothesized that the antigen-specific immune non-responsiveness to conventional tumor-associated antigens may be avoided by targeting tumor antigens that are induced after exposure to a cancer therapeutic or prevention agent as a mechanism of developing therapeutic resistance. Although there may be many potential antigens overexpressed in response to a cancer therapeutic or prevention agent, those antigens that are likely critical components of specific therapeutic resistance mechanisms would be attractive targets, as immunologic ablation of clones expressing such antigens should eliminate the clinical recurrence of therapy resistant tumor cells. One such antigen thought to be essential to therapeutic resistance is the estrogen receptor, ESR1. As proof of this concept in ER+ breast cancer, we have chosen to focus this approach on the recently uncovered resistance mutations to Estrogen Receptor alpha (ESR1).

We recently demonstrated that polyclonal antibodies induced by vaccination against receptors such as HER2 and HER3 can mediate profound receptor internalization and degradation, providing a therapeutic effect in vitro and in vivo (Ren et al., Breast Cancer Research 2012 14: R89 and International Patent Application No. WO 2013/110030, both of which are incorporated herein by reference in their entireties).

A vaccine composed of single or multiple forms of the ESR1 gene (Estrogen Receptor alpha) encoded by a platform that would elicit an immune response to the wild type or mutated epitopes of ESR1 is provided herein. In one embodiment, vaccines comprising one or more (or all) or portions of one or more (or all) of SEQ ID NOs: 1-3 and SEQ ID NO: 5 are provided. In the Examples, each vaccine contained a single ESR1 mutant, but more than one mutant can be included in a single vaccine. While others have utilized different approaches to target wild-type ESR1 through vaccination, our approach would selectively target forms of this gene that enable endocrine therapy resistance in tumors, i.e. mutant forms of ESR1 that allow the cancer cells to escape a therapeutic agent and continue growth. As such, we would expect that targeting these forms would prevent their emergence and would effectively prevent the development of resistance to endocrine therapies in ER+ breast cancer as well as other endocrine dependent cancers. Additionally, selectively targeting this specific mutant form of ESR1 would allow for effective tumor-specific anti-cancer activity mediated through immune targeting.

This invention would optimally be utilized through the inclusion of antigens encoded by the wild type and/or mutant forms of ESR1 gene (Y537N, Y537S, D538G, K303R and others), optimized forms of this gene (truncated, inactivated or otherwise), or specific combinations of peptide/epitopes of this gene in different immune stimulatory vector systems. The mutant forms of ESR1 gene (Y537N, Y537S, D538G, K303R) are provided as SEQ ID NOs; 1-3 and 5, respectively. Portions of these polypeptides may also be included in the vaccine. Suitably the portion included in the vaccine includes the mutation at the indicated amino acid. The portion included in the vaccine may include the mutation at only one of 537, 538 or 303 or may include small peptide epitopes comprising two of more of these mutations. The vaccine may include further peptides with additional mutations in the ESR1 protein in addition to those identified herein. The B and T cell epitopes being recognized after vaccination in the examples have not been identified, but those of skill in the art would expect the epitopes to be 6, 8, 10, 12, 14, 16, 18 or 20 amino acids in length. The Examples do suggest that the epitope includes the mutation at 537 for the vaccine containing SEQ ID NO:1 as the immune response generated after vaccination with the vaccine comprising SEQ ID NO: 1 did not recognize wild-type ESR1. The vaccines used in the Examples encompass larger polypeptides, but vaccines may include smaller portions of the ESR1 polypeptides than those provided herein. Suitably the vaccines include the region flanking the mutations at amino acid 537, 538 or 303 of the sequences and include at least 8, 10, 12, 14, 16, 18, 20 or more amino acids.

A polynucleotide encoding a polypeptide of SEQ ID NO: 1-3 or 5 or a portion thereof may be encompassed in a vaccine vector. Suitable vaccine vectors include, but are not limited to viral vectors such as adenoviral, fowlpox, vaccinia, VEE, etc., DNA-based vaccination vectors, or protein/peptide vaccination strategies. Liposomes or bacterial vaccine vectors may also be suitable. This immunotherapeutic platform could be used prior to the development of cancer types prior to the development of endocrine resistance, used in front line or adjuvant settings as a treatment for these cancers, and also as a preventative measure to prohibit the development and evolution of this signaling pathway as a resistance pathway.

The vaccines or vaccine vectors may include polynucleotides encoding additional polypeptides, such as HER3, HER2 or polypeptides of either of these comprising mutations such as those provided in SEQ ID NOs: 6-10 or any of the epitopes provided in International Publication No. WO2013/110030, which is incorporated herein by reference in its entirety. The vaccines or vaccine vectors may also include or be administered in conjunction with a checkpoint inhibitory immunomodulatory agent. The checkpoint inhibitory immunomodulatory agent may be an antibody antagonistic for CTLA-4 or PD1. In the Examples a PD1 antibody obtained from BioXCell called RMP1-14 and a CTLA-4 antibody from BioXCell called 9D9 were used. Other similar antibodies are commercially available or in clinical trials such as ipilimumab and nivolumab.

This would be easily distinguished from our and other prior approaches targeting wild-type ESR1 as the mutations to different portions of this gene render them insensitive to endocrine-targeted therapies with enhanced oncogenic potential. As such, vaccines targeting these mutant forms may elicit a different epitope repertoire for immune targeting and potentially a more significant anti-tumor effect by specifically targeting the development of endocrine resistance and specifically preventing it through immunoselective pressure.

Generation of resistance to cancer therapeutic or prevention agents is a common problem in the treatment of cancer or precancer and in several cases the mechanism of resistance to the therapeutic agent is known. Resistance is often the result of changes in gene expression (over-expression or blocked expression of a protein), change in the gene by mutation, or altered sequences by altered splicing or translocation or altered activation of a protein in the cells (over-activation or blocked activation of a protein).

In those cases where over-expression or over-activation of a protein, or a new sequence in the protein is responsible for increasing the resistance of the cancer or precancer cells to the therapeutic or prevention agent, we report a method for reducing the likelihood that the cancer or precancer will develop resistance to the cancer therapeutic or prevention agent. As used herein, resistance to a cancer therapeutic or prevention agent indicates that the cancer therapeutic or prevention agent is not as effective at inhibiting the growth of, or killing, cancer or precancer cells in response to the cancer therapeutic or prevention agent. The method may even block the development of resistance to the cancer therapeutic or prevention agent or may reverse resistance to the cancer therapeutic or prevention agent after it has developed. The methods include administering the cancer therapeutic or prevention agent and administering a vaccine to the subject in need of treatment for a cancer. The vaccine comprises a polynucleotide encoding a polypeptide whose expression or activation is correlated with or results in development of resistance of the cancer or precancer to the cancer therapeutic or prevention agent. The vaccines provided herein include an ESR1 polypeptide or a polynucleotide encoding an ESR1 polypeptide such as the polypeptide of SEQ ID NO: 2, 4, or 6.

The vaccine may be administered before, during or after treatment with a cancer therapeutic or prevention agent or may be administered simultaneously with the cancer therapeutic or prevention agent. The administration of the vaccine and the cancer therapeutic or prevention agent to the subject reduces the likelihood that the subject's cancer or precancer will develop resistance to the therapeutic or prevention agent as compared to a control subject with a similar cancer or precancer not administered the vaccine or as compared to the general likelihood of a population of subjects having the cancer or precancer. In some embodiments, the cancer or precancer in individuals administered both the vaccine and the therapeutic or prevention agent does not develop resistance to the cancer therapeutic or prevention agent and is treated. Alternatively, the growth of the cancer or precancer may be inhibited or the growth rate reduced. The administration of the vaccine and cancer therapeutic or prevention agent may also reverse resistance to the cancer therapeutic or prevention agent if the cancer or precancer is already resistant to the cancer therapeutic or prevention agent. In some embodiments, administration of the vaccine is sufficient to treat the cancer or inhibit the growth or kill the cancer. In other embodiments, the vaccine must be administered in conjunction with the cancer therapeutic or prevention agent or prior to development of resistance to the cancer therapeutic or prevention agent by the cancer.

The vaccine may include a polynucleotide encoding an ESR1 polypeptide. Three point mutations (4 mutant forms) of ESR1 associated with resistance to cancer therapeutic agents are provided as SEQ ID NOs: 1-3 and 5. The vaccine may comprise full-length ESR1 or portions thereof. For example, the vaccine may comprise only the epitopes identified in the examples or peptides comprising the mutations or deletions associated with resistance. Suitably the vaccine is capable of eliciting an immune response to ESR1 in a subject administered the vaccine. The immune response may be a B cell or T cell response. Suitably the immune response includes an antibody response directed to ESR1. The immune response may be a polyclonal antibody response in which multiple epitopes of ESR1 are recognized by antibodies. The immune response may recognize an epitope including the mutations such that after immunization the wild-type protein is not recognized or not recognized as strongly as the mutant form.

ESR1, as shown in SEQ ID NOs: 1-3 and 5, comprises mutations as indicated that lead to endocrine resistance. The mutations result in a unique sequence in the peptide and epitopes spanning these mutations can be identified and antibodies generated using the vaccines described herein. Those of skill in the art will appreciate that a vaccine including polynucleotides encoding only portions of full-length ESR1, i.e. antigenic epitopes, may be used in the vaccines described herein. Portions of the ESR1 including the mutation sites can be included in the vaccine. We have the following isolated ESR and ESR related peptides presented by MHC molecules on tumor cells.

TABLE 1 T cell epitopes derived from estrogen receptor related proteins: MCF10 (SEQ ID NO:) IQGNELEPL (11) A2 Estrogen receptor ESR1 MCF7 FMVLQVIKF(12) A24 nuclear receptor subfamily 1 group 1 member 3 isoform 15 LEMLEAKV (13) non estrogen-related receptor A2/A24 beta MDA EVFLPQRA (14) A2 estrogen-related receptor beta IFLNTEVSL (15) A24 estrogen receptor coactivator LTAEETDKI (16) A2/A24 estrogen receptor coactivator LTSSSIDPGIL (17) A2 estrogen receptor binding protein variant MLKHKRPLA (18) A2/A24 estrogen receptor alpha splice variant, partial TIVSLDAARR (19) A2 estrogen-responsive B box protein KGDEEKENN (20) non estrogen receptor-related A2/A24 protein LCVKAMILL (21) non estrogen receptor 2 (ER A2/A24 beta) variant MNQKLSPFM (22) non estrogen sulfotransferase A2/A24 RYKKLKVE (23) non estrogen-related receptor A2/A24 beta SKAKSLTDPS (24) non estrogen receptor binding A2/A24 protein variant WFGIKAPE (25) non estrogen receptor binding A2/A24 protein variant Any of these polypeptides individually or in combination may be used as ESR1 polypeptides in the vaccine described herein.

The vaccine may include a vaccine vector. The vaccine vector may be a bacterial, yeast, viral or liposomal vaccine vector. The vaccine may be a DNA vaccine as well and not include a vaccine vector. The vaccine vector may be an adenovirus, adeno-associated virus, fowlpox, vaccinia, viral equine encephalitis virus, venezuelan equine encephalitis virus or other viral vaccine vectors. One method for generating adenovirus vectors is provided in Luo et al., Nature Protocols, (2007) 2: 1236-1247, which is incorporated herein by reference. The vaccine vector may contain the ESR1 polynucleotide or portions thereof. The vaccine vector may contain the ESR1 polypeptide or portions thereof. The vaccine vector may express the ESR1 polypeptide or portions thereof. ESR1 polypeptide or portions thereof may be expressed on the surface or interior of the vaccine vector. ESR1 polynucleotide or portions thereof may be carried within the vaccine vector and the ESR1 polypeptide or portions thereof may be expressed only after vaccination. ESR1 polypeptides or portions thereof may be expressed as a fusion protein or in conjunction with adjuvants or other immunostimulatory molecules to further enhance the immune response to the polypeptide.

Methods of treating a cancer or precancer, or of reducing the likelihood of the cancer or precancer developing resistance to a cancer therapeutic or prevention agent, are also provided. The methods include administering the vaccine as described above to a subject having cancer or precancer. The subject may be any mammal, suitably a human, domesticated animal such as a dog or cat, or a mouse or rat. A cancer therapeutic or prevention agent may be administered concurrently with, before or after administration of the vaccine.

The cancer therapeutic or prevention agents may be any agent capable of treating the cancer or inhibiting growth of cancer cells. Suitable agents include those which target HER2, HER1/EGFR, HER3, estrogen receptor or IGF1R. The therapeutic agent may be trastuzumab, lapatinib, pertuzumab or another HER2 or estrogen receptor targeting therapeutic agent or it may be an EGFR targeting therapeutic agent such as cetuximab or erlotanib, or it may be an anti-estrogen, or an agent that prevents estrogen synthesis such as an aromatase inhibitor. We have previously demonstrated that a HER3 vaccine can treat a HER2 positive cancer when used in combination with a therapeutic agent targeting HER2. An ESR1 vaccine should work similarly and the mutations provide unique sites for vaccination to differentiate cancer or precancer cells from normal cells. Cancer cells often develop resistance to therapeutic agents. Addition of vaccination with an ESR1 vaccine or passively transferred polyclonal antibodies specific for ESR1 will result in blocking resistance, inhibit cancer cell growth and result in treatment of the cancer.

Suitably the vaccinated subject develops an immune response to the mutated form of ESR1 in response to administration of the vaccine. The immune response may be an antibody or T cell immune response. For example the immune response may include antibody-dependent cellular cytotoxicity, polyclonal antibody response, complement dependent cellular cytotoxicity, cellular cytotoxicity, disruption of ligand binding, disruption of dimerization, mimicking ligand binding causing internalization of ESR1, or degradation of ESR1. The immune response may comprise an antibody response directed to at least a portion of ESR1, suitably a portion including the mutations. The immune response may be specific for a T cell or B cell epitope flanking or encompassing the mutations in SEQ ID NO: 1-3 or 5 or regions flanking the mutations in ESR1.

Reduction of the development of resistance can be measured in several ways. The resistance of the vaccinated subject may be compared to a similar subject that was not vaccinated as in the Examples. Alternatively, the reduction may be measured based on statistics generated regarding the likelihood of an individual being treated with the therapeutic agent to develop resistance versus that of individuals treated with the therapeutic agent and vaccinated with ESR1. The reduction in the likelihood of resistance of the cancer may also be measured by measuring the level of ESR1 expression on the surface of cancer cells. ESR1 expression is reduced on cancer cells after effective administration of the vaccine. The effectiveness of the vaccine in treating the cancer or reducing the likelihood of resistance can be measured by tracking the growth of the tumor or the growth rate of the tumor or cancer cells. A decrease in tumor size or in the rate of tumor growth is indicative of treatment of the cancer.

The cancer may be selected from any cancer capable of developing resistance to a therapeutic agent by increasing expression or activation of a protein by the cancer cells. In particular the cancer may be any cancer capable of developing resistance to a therapeutic agent which targets a HER family tyrosine kinase, suitably HER2, HER3, or EGFR or the estrogen receptor, suitably anti-estrogens. The cancer may develop resistance by increasing the expression of ESR1, mutating ESR1 or deleting a portion of ESR1 to avoid susceptibility to the therapeutic agent. Suitably the cancers are selected from breast, prostate, lung, ovarian, colon, rectal, pancreas, bladder, head and neck or liver cancers or precancers. The resistance may be due to a single or multiple changes, and the vaccine can target one or more of these changes, and/or include multiple antigens likely found in resistance cells, but not necessarily in all resistance cells.

Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor in the subject, reducing progression of a cancer to a more aggressive form (i.e. maintaining the cancer in a form that is susceptible to a therapeutic agent), reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with cancer or at risk of developing cancer or facing a cancer recurrence. Treatment includes improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay in the onset of symptoms or slowing the progression of symptoms, etc.

Co-administration, or administration of more than one composition (i.e. a vaccine and a therapeutic agent) to a subject, indicates that the compositions may be administered in any order, at the same time or as part of a unitary composition. The two compositions may be administered such that one is administered before the other with a difference in administration time of 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more.

An effective amount or a therapeutically effective amount as used herein means the amount of a composition that, when administered to a subject for treating a state, disorder or condition is sufficient to effect a treatment (as defined above). The therapeutically effective amount will vary depending on the compound, formulation or composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated.

The compositions (i.e. the vaccines and the therapeutic agents or checkpoint inhibitory agents) described herein may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, or transmucosal absorption. Thus the compositions may be formulated as an ingestable, injectable, topical or suppository formulation. The compositions may also be delivered with in a liposomal or time-release vehicle. Administration of the compositions to a subject in accordance with the invention appears to exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the composition or compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compositions described herein and of a known agent, such as by means of an appropriate conventional pharmacological or prophylactic protocol.

The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual prophylactic or treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compositions will reduce the growth of the cancer at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more as compared to no treatment or treatment with only the therapeutic agent. It is specifically contemplated that pharmaceutical preparations and compositions may palliate, block further growth or alleviate symptoms associated with the cancer without providing a cure, or, in some embodiments, may be used to cure the cancer and rid the subject of the disease.

The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition is administered, the effective dosage amounts correspond to the total amount administered. The compositions can be administered as a single dose or as divided doses. For example, the composition may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks.

The vaccine vector may be administered one time or more than one time to the subject to effectively boost the immune response against ESR1. If the vaccine is provided as a vaccine vector, the vaccine vector may be administered based on the number of particles delivered to the subject (i.e. plaque forming units or colony forming units). The subject may be administered 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷ or 10⁶ particles.

The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

Examples Materials and Methods

Viral Vectors:

Mutations in the ESR1 plasmid were generated by site-directed mutagenesis of a pENTR221-ESR1 plasmid obtained from the Orfeome (Dharmacon) to create ESR1 mutants Y537N, Y537S, D538G, and K303R. See SEQ ID Nos: 1-3 and 5, respectively. These genes were cloned into adenoviral vectors, which were then generated as previously described¹. These genes were also cloned into lentiviral vectors (LX301 from Addgene) and used to generated cell lines with stable expression of these genes as previously described².

Cell Lines:

Breast epithelial cell lines MM3MG, NMuMG, and MCF-7 were obtained from the American Tissue Culture Collection (ATCC), and were maintained according to ATCC recommendations. These lines were tested for Mycoplasma and DNA fingerprinted at the Duke Cell Culture Facility.

In Vitro Assays:

Proliferation of stable cells was determined by MTT assay using 5,000 cells/well over the course of 3 days (against control counterparts) in 96-well plates. MTT growth assessments were done using a Bio-Rad plate reader after cell solubilization in DMSO. Soft agar assays for stable expression were done as described. Briefly, 50,000 cells/well were plated in 0.3% soft agar (on a base of 0.6% soft agar) and allowed to grow for a period of 2 wk in DMEM with 10% FBS. At the end of this time, colonies of >15 cells were counted and scored. In experiments using inducible expression systems, Dox was added to the media at a concentration of 2 μg/ml (replaced weekly). Wound Scratch Assays were performed using p1000 tips, washing wounded plates with PBS (2×) and applying media before staining with Crystal Violet at 16 hours post-wounding. Pictures were taken using an Olympus 1X73 using a 10× magnification objective. Luciferase Assays were performed by co-transfecting an luciferase reporter plasmids (SABiosciences) along with a CAG-LacZ control (Addgene) into modified 293T cells in estrogen-free conditions (phenol-red free media with charcoal stripped FBS) and assessing pathways signaling at 24 or 48 hpt using standard techniques with Luminometer (GloMax 96-well, Promega, Madison Wis.).

Animal Experiments:

Experiments using BALB/c and SCID-Beige mice (obtained from Jackson Labs) were done in accordance with Duke Institutional Animal Care and Use Committee-approved protocols. For tumor vaccine experiments, BALB/c mice (Jackson Labs) were implanted with MM3MG modified cellsR2d16 tumors and genotyped by PCR as previously described¹. In immune competent (BALB/c) and immuno-deficient animals (SCID-beige), stable cells were injected s.c. into the flank of SCID-beige mice (at indicated cells/animal) and measured as indicated. Tumor measurements were made using calipers and volumes calculated using the formula [v=width×width×(length/2)] whereas statistical differences were calculated using a mixed effects regression model using autoregressive covariance.

ELISPOT and ELISA Assays:

Immunogenicity experiments involved footpad injection of Ad-ESR-WT, ESR1-mutant, and Ad-GFP vectors (2.6×10¹⁰ particles/mouse) in BALB/c animals. Fourteen days post-injection, mice were euthanized and splenocytes and sera were collected for analysis. IFN-γ ELISPOT assays (Mabtech Inc.) were done according to the manufacturer's instructions using overlapping ESR1 peptide mixes (2.6 μg/mL; BD Biosciences) as stimulating antigens and HIV-irrelevant overlapping peptide mixes as negative controls (BD Biosciences). Phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μg/mL) served as a positive control for splenocyte responsiveness. Antibodies were assessed using a sandwich-based ELISA method using ESR1 protein (10 ug/ml, Thermo-Fisher) as previously described using LacZ⁵.

Results:

To initially validate the importance of the identified ESR1 mutations, we first investigated their ability to elicit canonical estrogen-dependent signaling in the absence of exogenous estrogen stimulation. To test differences in ESR1 estrogen-dependent signaling, we generated 293T cells (which do not express detectable levels of ESR1) expressing ESR-WT, Y537N, Y537S, D538G, and K303R. Using a highly sensitive canonical ERE-pathway luciferase-based signaling reporter system, we then determined how estrogen-free and estrogen-induced ERE pathways were affected by ESR1-WT or mutant expression. We found that ERE signaling was strongly activated in ESR1-WT expressing cells when stimulated with estrogen in comparison to ESR1-WT expressing non-stimulated cells or control cells stimulated with estrogen (FIG. 1). More importantly, all mutant forms of ESR1 strongly induced the ERE pathway in the absence of estrogen (FIG. 1). To get a broader view of potential pathways affected by these mutants, we utilized a high throughput 45-pathways reporter array (SABiosciences) for both estrogen receptor mutation classes (represented by ESR1-Y537S and K303R). These experiments revealed that both classes of estrogen receptor mutants elicited ERE signaling, but also PR and RAR signaling pathways (FIGS. 2 and 3). Having identified the three critical pathways activated by estrogen receptor mutants, we then validated their activity in ESR1-WT expressing cells stimulated with estrogen, as well as in the other ligand-binding domain mutants (ESR1-Y537N and ESR1-D538G) (FIG. 4-5). Our results demonstrate that these pathways are stimulated by ESR1-WT only in the presence of estrogen, but are constitutively active in mutant ESR1 expressing lines in the absence of estrogen stimulation.

Having demonstrated that these mutants can confer robust ESR1 canonical signaling that mimics estrogen stimulation, we next wanted to see if these receptors could compensate for loss of estrogen stimulation in ER+ breast cancer. To test this, we constructed a series of MCF-7 cells with expression of wild-type or mutated ESR1 and implanted these cells into male mice (low estrogen levels) without any exogenous estrogen stimulation. As a control, we implanted both MCF-7 control cells (high ESR1) and MCF-7-ESR1-Y537N cells into female mice supplemented with exogenous estrogen. As predicted exogenous estrogen supplementation allowed for 100% tumor growth, while estrogen starvation resulted in strongly reduced tumor growth of control cells in 13% of mice and no control tumor growth in 87% of mice. This was in direct contrast to ESR1 mutant expressing cells which formed tumors in 80-100% of mice without exogenous estrogen supplementation (FIG. 6). However, ESR1-Y537N tumor growth in low estrogen conditions was somewhat slower non-supplemented mice, thus potentially demonstrating an effect from ESR1-WT present in MCF-7 cells. Collectively, these results demonstrate that these identified ESR1 mutants can compensate for suppression of estrogen in ER+ breast cancer in vivo, suggesting their efficacy as anti-estrogen therapies resistance mechanisms.

To determine if we could successfully immunologically target these mutated versions of ESR1 in a breast cancer setting, we first needed to establish a mouse model system of ER+ breast cancer. This model system would ideally have some form of transformation driven estrogen dependence whereby estrogen signaling was driven by these mutant versions of ESR1. To establish this model system, we first transduced MM3MG mammary breast epithelial cells with lentiviral vectors to stably express the various forms of ESR1. This mammary epithelial line was only weakly transformed (data not shown), expressed low endogenous levels of ESR1, and from a BALB/c background which would allow for immune studies to be performed in syngeneic BALB/c mice. After establishing these cell lines for each class of ESR1 mutant with a wild-type ESR1 control, we first confirmed that ERE signaling occurred in control lines after estrogen stimulation and in an ESR1 mutant line without estrogen stimulation (FIG. 7). To determine if these mutants had oncogenic capacity in these cells, we ascertained proliferation, anchorage-independent growth in soft agar, as well tumor formation and growth in vivo. Using MTT-based assays, we found that these mutants had no effect on proliferation (FIG. 8), although we did observe a significant increase in anchorage-independent growth when ESR1-Y537N was expressed in soft agar assays (FIG. 9). Finally, we injected ESR1-WT, ESR1-K303R, and ESR1-Y537N to determine the effect of ESR1-WT or mutated ESR1 on tumor growth in vivo. Notably, we found that both mutant ESR1 expressing lines grew more rapidly in these mice, demonstrating an oncogenic capacity of mutant estrogen signaling in these cells. Collectively, these results demonstrate that these mutants can confer estrogen-independent canonical signaling through several different pathways and that these mutant ESR1 can enhance murine mammary cell tumor growth in vivo.

Recent clinical studies have indicated that ESR1 mutations can be responsible for tamoxifen resistance in patients, which is supported by our findings using ESR1 mutant expressing cell lines. We hypothesized that immunologic targeting of these genes may prevent the development of resistance and potentially be a therapy for ER+ breast cancer. As such, we wished to explore the capacity of a vaccine targeting these oncogenic mutant forms of ESR1 to elicit anti-tumor responses. As a preliminary step, we took advantage of our adenoviral vector platform, which we had previously demonstrated to be capable of eliciting strong anti-tumor immunity against multiple Tumor Associated Antigens, including HER2. Using this platform, we constructed adenoviral vectors encoding wild-type and mutant forms of ESR1. After constructing and purifying these vectors, we ascertained their ability to elicit ESR1-specific immunity in BALB/c mice. Using a ESR1-specific ELISPOT assay, we determined that vaccination with ESR1 or mutant ESR1 genes strongly elicited significant T-cell mediated immunity to ESR-specific epitopes (FIG. 11), while a ESR1-specific ELISA assay demonstrated significant ESR1 specific antibody responses (FIG. 12).

Having thus demonstrated these vaccines were capable of eliciting B-cell and T-cell ESR1-specific immunity, we next sought to determine if vaccination against a constitutively active oncogenic ESR1 mutant could significantly retard tumor growth. Having developed ESR1-mut transformed breast cancer lines capable of growing in immunocompetent transgenic animals, we next implanted these cells or ESR1-WT expressing counterparts into animals and tested if anti-ESR1 responses elicited by a preventative vaccination (2 weeks prior to injection) could retard ESR1-mediated growth. Our results demonstrated that an Ad-ESR1-Y537N vaccine formulation could significantly suppress ESR1-Y537N mediated tumor growth, but surprisingly did not affect MM3MG-ESR1-WT tumor growth (FIG. 13).

Additionally, we found that after a control Ad-GFP vaccination, MM3MG-ESR1-Y537N cells grew much more rapidly than MM3MG-ESR1-WT cells, thus demonstrating that an active form of ESR1 also enhanced growth in an immunocompetent setting. This was confirmed by ELISPOT assays that indicated Ad-ESR1-Y537N could elicit ESR1-specific T-cell responses in vaccinated tumor-bearing animals (FIG. 14). These results suggest that therapies targeting ESR1 mutants can have a significant immunologic impact on tumor growth in cancers driven by specific oncogenic mutations. As such, we expect that the use of checkpoint inhibitors in combination with our vaccine will allow for significant and sustained immune responses to critical oncogenic drivers and may prevent the development of resistance mediated by ESR1 mutation.

-   1. Hartman, Z. C. et al. An adenoviral vaccine encoding full-length     inactivated human Her2 exhibits potent imnunogenicty and enhanced     therapeutic efficacy without oncogenicity. (Clin. Cancer Res. 16,     1466-1477 (2010). -   2. Hartman, Z. C. et al. Growth of triple-negative breast cancer     cells relies upon coordinate autocrine expression of the     proinflammatory cytokines IL-6 and IL-8. Cancer Res 73, 3470-80     (2013). -   3. Hartman, Z. C. et al. HER2 overexpression elicits a     proinflammatory IL-6 autocrine signaling loop that is critical for     tumorigenesis. Cancer Res. 71, 4380-4391 (2011). -   4. Kershaw, M. H. et al. Gene-engineered T cells as a superior     adjuvant therapy for metastatic cancer 1. J. Immumol. 173, 2143-2150     (2004). -   5. Hartman, Z. C. et al. Ligand-independent toll-like receptor     signals generated by ectopic overexpression of MyD88 generate local     and systemic antitumor immunity. Cancer Res. 70, 7209-7220 (2010). -   6. Early Breast Cancer Trialists' Collaborative, G. et al. Relevance     of breast cancer hormone receptors and other factors to the efficacy     of adjuvant tamoxifen: patient-level meta-analysis of randomised     trials. Lancet 378, 771-84 (2011). -   7. Baum, M. et al. Anastrozole alone or in combination with     tamoxifen versus tamoxifen alone for adjuvant treatment of     postmenopausal women with early breast cancer: first results of the     ATAC randomised trial. Lancet 359, 2131-9 (2002). -   8. Nabholtz, J. M. et al. Anastrozole is superior to tamoxifen as     first-line therapy for advanced breast cancer in postmenopausal     women: results of a North American multicenter randomized trial.     Arimidex Study Group. J Clin Oncol 18, 3758-67 (2000). -   9. Ignatiadis, M. & Sotiriou, C. Luminal breast cancer: from biology     to treatment. Nat Rev Clin Oncol 10, 494-506 (2013). -   10. Yerushalmi, R. et al. Tumor markers in metastatic breast cancer     subtypes: frequency of elevation and correlation with outcome. Ann     Oncol 23, 338-45 (2012). -   11. Yerushalmi, R. et al. Patterns of relapse in breast cancer     changes over time. Breast Cancer Res Treat 120, 753-9 (2010). -   12. Kennecke, H. et al. Metastatic behavior of breast cancer     subtypes. J Clin Oncol 28, 3271-7 (2010). 

1. A vaccine comprising a vaccine vector comprising a polynucleotide encoding mutant ESR1 polypeptide or a portion thereof.
 2. The vaccine of claim 1, wherein the mutant ESR1 polypeptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 or portions of any of SEQ ID NO: 1-3 or
 5. 3. The vaccine of claim 1, wherein the mutant ESR1 polypeptide consists of at least one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO:
 5. 4. The vaccine of claim 1, wherein the portion of the ESR1 polypeptide comprises a mutation in ESR1.
 5. The vaccine of claim 1, further comprising a HER3 polypeptide or HER2 polypeptide or a mutant form of HER3 or HER2.
 6. (canceled)
 7. The vaccine of claim 1, wherein the vaccine vector comprises the mutant ESR1 polypeptide.
 8. The vaccine of claim 7, wherein the vaccine vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), fowlpox, vaccinia virus, and Venezuelan equine encephalitis virus.
 9. (canceled)
 10. The vaccine of claim 1, further comprising a checkpoint inhibitor immunomodulatory agent.
 11. The vaccine of claim 10, wherein the checkpoint inhibitor immunomodulatory agent is a CTLA-4 or PD1 antagonistic antibody.
 12. A method of treating a cancer or precancer or of reducing the likelihood of the cancer developing resistance to a cancer therapeutic or prevention agent comprising administering the vaccine of claim 1 to a subject having the cancer or precancer, wherein administration of the vaccine to the subject treats the cancer or precancer, reduces the likelihood of the cancer or precancer developing resistance to the cancer therapeutic or prevention agent or reverses resistance of the cancer or precancer to the cancer therapeutic or prevention agent.
 13. The method of claim 12, wherein the cancer comprises a mutation in ESR1.
 14. The method of claim 12, wherein the vaccine is administered concurrently with, before or after administration of the cancer therapeutic or prevention agent.
 15. The method of claim 14, wherein the cancer therapeutic or prevention agent is an agent targeting HER2, HER1, estrogen receptor, EGFR, or IGF1R.
 16. The method of claim 12, wherein the vaccine is administered concurrently with, before or after administration of a checkpoint inhibitor immunomodulatory agent.
 17. The method of claim 16, wherein the checkpoint inhibitor immunomodulatory agent is a CTLA-4 or PD1 antagonistic antibody.
 18. The method of claim 12, wherein the cancer or precancer is selected from a breast, prostate, lung, ovarian, colon, rectal, pancreas, bladder, head and neck or liver cancer or precancer.
 19. The method of claim 12, wherein the subject develops an immune response to ESR1.
 20. The method of claim 19, wherein the immune response comprises an antibody response or a T cell mediated response.
 21. The method of claim 19, wherein the immune response includes at least one of antibody-dependent cellular cytotoxicity, polyclonal antibody response, complement dependent cellular cytotoxicity, cellular cytotoxicity, disruption of ligand binding, disruption of dimerization, mimicking ligand binding causing internalization of ESR1, or degradation of ESR1.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. The method of claim 12, wherein the cancer therapeutic or prevention agent is selected from trastuzumab, lapatinib, cetuximab, pertuzumab and erlotanib. 